Ca2+/calmodulin regulation of putrescine uptake in cultured gastrointestinal epithelial cells.
Ca2+/calmodulin regulation of putrescine uptake in cultured gastrointestinal epithelial cells.
复制标题
Ca2 /钙调蛋白对培养的胃肠上皮细胞中腐胺摄取的调节。
DOI：
10.1152/ajpcell.1992.262.6.c1356
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
Seidel,ER
中科院分区：
文献类型：
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作者：
Groblewski,GE;Hargittai,PT;Seidel,ER
调节腐胺摄取小肠隐窝细胞系，IEC-6细胞，进行了检查。[14 C]腐胺的摄取在整个正常的生长曲线进行测量，并被发现是负相关的增长。低和高细胞密度下的动力学分析显示，融合细胞中的摄取抑制是由于摄取Vmax降低5倍，分别为199.5 pmol. 10（5）cells-1.h-1 vs. 43.1 pmol. 10（5）cells-1.h-1。三种胃肠激素，胃泌素，促胰液素和胆囊收缩素，产生部分抑制[14 C]腐胺摄取。相反，用5%胎牛血清处理静止细胞以刺激生长并不影响摄取。采用钙离子敏感的荧光探针Fura-2，用显微荧光法测定腐胺摄取对细胞内游离钙离子浓度（[Ca 2 +]i）的影响。基础[Ca 2 +]i计算为112 nM，加入10 μ M腐胺后迅速增加至313 nM。使用细胞内Ca 2+缓冲液1，2-双（2-氨基苯氧基）乙烷-N，N，N '，N'-四乙酸乙酰氧基甲酯防止[Ca 2 +]i升高，使[14 C]腐胺摄取降低至对照值的29.5 +/- 5.3%。~（45）Ca ~（2+）通量实验和在0 Ca ~（2+）和0.5 mM EDTA中的运输测量表明，在腐殖酸摄取过程中，细胞内钙源被动员。最后，使用假定的钙调素拮抗剂N-（6-氨基己基）-5-氯-1-萘磺酰胺引起[14 C]腐胺摄取的剂量依赖性抑制，50%抑制浓度约为7 μ M。这些数据表明，腐胺在IEC-6细胞的摄取可能是由钙/钙调素依赖性机制。
Regulation of putrescine uptake in a small intestinal crypt cell line, IEC-6 cells, was examined. Uptake of [14C]putrescine was measured throughout a normal growth curve and was found to be inversely related to growth. Kinetic analysis at low and high cell density revealed the inhibition of uptake in confluent cells was due to a five-fold reduction in Vmax of uptake, 199.5 vs. 43.1 pmol.10(5) cells-1.h-1, respectively. Three gastrointestinal hormones, gastrin, secretin, and cholecystokinin, produced partial inhibition of [14C]putrescine uptake. Conversely, treatment of quiescent cells with 5% fetal bovine serum to stimulate growth did not affect uptake. Influence of putrescine uptake on free ionized intracellular Ca2+ ([Ca2+]i) was measured by microspectrofluorometry using the Ca(2+)-sensitive fluoroprobe fura-2. Basal [Ca2+]i was calculated to be 112 nM and increased rapidly to 313 nM upon addition of 10 microM putrescine. Preventing the rise in [Ca2+]i using an intracellular Ca2+ buffer, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, decreased [14C]putrescine uptake to 29.5 +/- 5.3% of control values. 45Ca2+ flux experiments and measurement of transport in 0 Ca2+ and 0.5 mM EDTA suggested an intracellular source of calcium was mobilized during putrescineuptake. Finally, use of the putative calmodulin antagonist N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide caused a dose-dependent inhibition of [14C]putrescine uptake with 50% inhibitory concentration of approximately 7 microM. These data suggest that putrescine uptake in IEC-6 cells may be regulated by a Ca2+/calmodulin-dependent mechanism.